Model Systems to Study HIV

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Models Systems to Study HIV
Emily L. Lowe, Ph.D.
Microbiology, Immunology and
Molecular Genetics
UCLA
Models Systems to Study HIV:
In vitro and In vivo
• In vitro: Cell lines, primary cells
• In vivo: Humanized mice
• In vivo: Non-human Primates
Models Systems to Study HIV:
In vitro and In vivo
• Genetically modified viruses
– Modify the envelope to allow entry
into more (or less) cell types
– Add a fluorescent protein to enable
better visualization
• Green fluorescent protein (GFP), green
• mCherry, red
• SIV (Simian Immunodeficiency
Virus)
• SHIV: chimeras of HIV and SIV
Yu et al. HIV traffics through a specialized,
surface-accessible intracellular compartment
during trans-infection of T cells by mature
dendritic cells. PLoS Pathog. 4 (8). 2008.
e1000134.
In vitro Models to Study HIV
• In vitro: Taking place in a test tube, culture
dish or elsewhere OUTSIDE a living organism
• Excellent tools for simple questions with
limited variables
– Best for testing more focused approaches
•
•
•
•
Cells are easily manipulated
Cost efficient
Scalable
Excellent for initial drug screens
Types of In vitro Models
• Cancer cell lines are IMMORTALIZED
– Can be expanded in culture for long periods of time
– Are easily manipulated to express (or not) CD4, CCR5
and/or CXCR4 as well as host restriction factors
• “Primary cells” come from people or animal
subjects
– Can purified to look at single or multiple populations
– Can only be maintained in culture for short periods
– Can be manipulated but not as easily as cancer cell
lines
Examples of in vitro applications
•
•
•
•
•
To see HIV virions
To see an HIV infected cell infect another cell
To see where HIV goes in a cell
To see what HIV interacts with in a cell
Identify latency inducers
In vitro study to see HIV virions
Cryofluorescence Light Microscope
170X
3500X
50,000X
June et al., Direct Visualization of HIV-with Correlative Live-Cell Microscopy and
Cry-Electron Tomography. Structure. 19 (11). 2011. 1573-1581.
In vitro study to see HIV virions
June et al., Direct Visualization of HIV-with Correlative Live-Cell Microscopy and
Cry-Electron Tomography. Structure. 19 (11). 2011. 1573-1581.
In vitro study to see an HIV infected
cell infect another cell
Dr. Thomas Huser’s Lab
http://cbst.ucdavis.edu/research/hiv-transmission-visualization
In vitro study to see where HIV goes in
a cell it has infected
Campbell et al., Visualization of a proteasome-independent intermediate during
restriction of HIV-1 by rhesus TRIM5alpha. J. Cell Biol. 180 (3). 2008. 549-561
In vitro study to see where HIV goes in
a cell it has infected
Green = HIV
Red = “body”
Campbell et al., Visualization of a proteasome-independent intermediate during
restriction of HIV-1 by rhesus TRIM5alpha. J. Cell Biol. 180 (3). 2008. 549-561
In vitro study to see what HIV interacts
with inside a cell
Red = host restriction factor
Green = HIV
Orange = interaction!!!
Campbell et al., Visualization of a proteasome-independent intermediate during
restriction of HIV-1 by rhesus TRIM5alpha. J. Cell Biol. 180 (3). 2008. 549-561
In vitro
study to
identify
latency
inducers
Kim et al., Recruitment of THIIH to the HIV LTR is a rate-limiting step in the
emergence of HIV from latency. EMBO J. 25. 2006. 3596-3604.
In vitro studies have given us a lot but…
• One major limitation to vaccine and
therapeutic cure research has been the lack of
an animal model that recapitulates all of the
salient features of HIV-1 infection in humans.
• In vivo: Experimentation using a whole, living
organism
In vivo Models to Study HIV
• When considering species other than human as
models for HIV-1 infection, the cellular proteins
of the species must support viral replication.
• Humanized mice
• Non-human primates
In vivo Models to Study HIV:
Humanized Mice
Most importantly, these animals CAN be infected
with HIV!
• Develop systemic viremia
• CD4 T cell loss
Akkina, R. New generation humanized mice for virus research: comparative aspects and future prospects. Virology.
435 (1). 2013. 14-28.
In vivo Models to Study HIV:
Humanized Mice
• Stem cells can be genetically manipulated (gene
therapy)
• Not terribly cost effective
– Surgery requires technical skill
– Cannot be bred
– Special housing/handling due to immune compromised
status
• Scalable allows for multiple “n”
– Can make up to 30 mice per tissue pair
– Excellent for initial drug-toxicity/efficacy screens
• IV and mucosal routes of infection possible
• Non-hematopoietic cells (lung, intestine mucosa)
cannot be studied in organ systems
In vivo Models to Study HIV:
Non-human primates
• Many species of African monkeys and apes are
natural hosts for SIV, but generally do not
develop disease as a consequence of
infection.
Natural Hosts (African)
SIVagm
African green monkey
©2011 by Cold Spring Harbor Laboratory Press
Sharp P M , and Hahn B H Cold Spring Harb Perspect Med
2011;1:a006841
In vivo Models to Study HIV:
Non-human primates
• By contrast, infection of Asian macaques,
which are NOT natural hosts for primate
lentiviruses, with certain strains of SIV results
in high viral loads, progressive CD4+ T cell
depletion and opportunistic infections.
Natural Hosts (African)
©2011 by Cold Spring Harbor Laboratory Press
Non-natural Hosts (Asian)
Sharp P M , and Hahn B H Cold Spring Harb Perspect Med
2011;1:a006841
In vivo Models to Study HIV:
Non-human primates and SIV
HIV-1 group M, which
is the most prevalent
HIV strain, jumped
from chimpanzees
into humans.
SIVcpz
HIV-1
http://www.awf.org/content/wildlife/detail/chimpanzee
HIV-2 originated in sooty
mangabeys and is
responsible for fewer
infections than HIV-1
http://pin.primate.wisc.edu/factsh
eets/entry/sooty_mangabey
SIVsmm
HIV-2 SIVmac
In vivo Models to Study HIV:
Non-human primates and SIV
• SIV: Simian Immunodeficieny Virus
– First isolated at the New England Primate Research
Center, Massachusetts, from rhesus macaques with a
transmissible form of immunodeficiency characterized
by opportunistic infections and tumours
– Later traced to an outbreak of lymphoma in the 1970s
among macaques that were housed at the California
National Primate Research Center, California
– Monkeys might have received tissues from SIVinfected sooty mangabeys during experiments aiming
to develop a non-human primate model for prion
disease
In vivo Models to Study HIV:
Non-human primates and SHIVs
• SHIV
– Simian immunodeficieny virus containing HIV
sequences/elements
– HIV-based vaccines cannot be tested with SIV
– SIV is not sensitive to many drugs that inhibit HIV-1
– SIV uses CD4 and CCR5 (like HIV-1) but my also use other
co-receptors complicating testing of entry inhibitors
• Env-SHIV
• RT-SHIV
• stHIV-1
In vivo Models to Study HIV:
Non-human primates and SHIVs
Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio. 10. 2012. 852-867.
In vivo Models to Study HIV:
Non-human primates and SHIVs
Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio. 10. 2012. 852-867.
In vivo Models to Study HIV:
Non-human primates and SHIVs
Pig tailed macaque
Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio. 10. 2012. 852-867.
In vivo
Models to
Study HIV:
Non-human
primates
Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio. 10. 2012. 852-867.
• Most commonly used
• Pathogenesis similar to
HIV-1
• High viral loads
• Progressive depletion of
mucosal or peripheral
CD4+ T cells
• Destruction of lymph
node architecture
• Progress AIDS faster (1-2
yrs)
Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio. 10. 2012. 852-867.
• Next most commonly
used
• Lower viral loads and less
CD4+ T cell depletion
• Progress AIDS faster
(within 42 weeks)
Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio. 10. 2012. 852-867.
• Least used
• SIV strains are less
pathogenic (probably
because they are
passaged in Rhesus
macaques)
Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio. 10. 2012. 852-867.
In vivo Models to Study HIV:
Non-human primates
• Stem cells can be genetically manipulated (gene
therapy)
• Cost prohibitive
• Primate research contains ethical controvery
• Not scalable
• IV and mucosal routes of infection possible
• All tissues match (non-hematopoietic and immune)
allowing for organ systems to be studied
Models to study HIV
Cats and Feline Immunodeficiency Virus
Wongsrikeao et al., Antiviral restriction factor transgenesis in the domestic cat.
Nature Methods. 8. 2011. 853-859.
Thank you!
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